Title:	Secretion signal vectors
Document Type and Number:	United States Patent 7071172
Link to this Page:	http://www.freepatentsonline.com/7071172.html
Abstract:	The present invention provides delivery vectors for transferring a nucleic acid sequence to a cell in vitro, ex vivo or in vivo. The delivery vector comprises a segment encoding a secretory signal peptide. In embodiments of the invention, the delivery vector is an adeno-associated virus (AAV) vector. In other embodiments, the secretory signal peptide is a fibronectin secretory signal peptide (including variations and modifications, thereof). The delivery vectors of the invention may further comprise a heterologous nucleic acid sequence encoding a polypeptide of interest for transfer to a target cell, where the polypeptide of interest is operably associated with the secretory signal. Also disclosed are methods of transferring a nucleic acid of interest to a cell using the delivery vectors of the invention.
Inventors:	McCown, Thomas J.; Haberman, Rebecca P.;
Application Number:	425328
Filing Date:	2003-04-29
Publication Date:	2006-07-04
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Assignee:	The University of North Carolina at Chapel Hill (Chapel Hill, NC)
Current Classes:	514 / 44 , 424 / 233.1, 435 / 320.1, 435 / 455, 435 / 456, 536 / 23.1, 536 / 23.2, 536 / 23.5, 536 / 23.51, 536 / 24.1
International Classes:	A61K 31/70 (20060101); A61K 39/23 (20060101); C07H 21/04 (20060101); C12N 15/86 (20060101)
Field of Search:	435/320.1 536/23.4,23.5 434/93.2
US Patent References:	5342762 August 1994 Mosher et al. 5618677 April 1997 Ni et al. 6040172 March 2000 Kaplitt 6180613 January 2001 Kaplitt et al. 6365394 April 2002 Gao et al.
Foreign Patent References:	WO 92/15015 Sep., 1992 WO
Other References:	Watson (Nucl. Acids Res. 12(13): 5145-5164). cited by examiner . Patel et al (EMBO 6:2565-2572, 1987). cited by examiner . Yurchenko et al (J. Biol. Chem. 268(11): 8356-8365, 1993). cited by examin- er . Schwarzbauer (J. Cell. Biol. 113(6): 1463-1473, 1991). cited by examiner . GenBank Accession No. NM.sub.--002026 (Nov. 15, 2001). cited by examiner . GenBank Accession No. Q91740 (Jul. 15, 1999). cited by examiner . DeSimone et al (Dev. Biol. 149 (2), 357-369 (1992). cited by examiner . Samulski et al (J. Virol. 63(9): 3822-3828, 1989). cited by examiner . D'Ercole et al (Progress in Growth Factor Research 6(2-4): 1995), abstract only. cited by examiner . During et al., "Glucagon-like peptide-1 receptor is involved in learning and neuroprotection," Abstract, Nat. Med. (Aug. 17, 2003). cited by other . Freese et al., "Direct Gene Transfer Into Human Epileptogenic Hippocampal Tissue with an Adeno-Associated Virus Vector: Implications for a Gene Therapy Approach to Epilepsy," Epilepsia 38(7): 759-766 (1997). cited by other . Goss et al., "Herpes Simplex-Mediated Gene Transfer of Nerve Growth Factor Protects Against Peripheral Neuropathy in Streptozotocin-Induced Diabetes in the Mouse," Diabetes 51 2227-2232 (Jul. 2002). cited by other . Haberman et al., "Inducible long-term gene expression in brain with adeno-associated virus gene transfer," Gene Therapy 1604-1611 (1998). cit- ed by other . Haberman et al., "Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure Sensitivity," Molecular Therapy 6(4) 495-500. cited by other . Haberman, et al., "Regulated Suppression of Focal Seizure Sensitivity by Adeno-Associated Virus (AAV) Vector-Dependent Galanin Secretion," Program No. 619.10 Abstract Viewer/Itinerary Planner Washington, DC: Society for Neuroscience (2002). cited by other . Huber et al., "Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy," PNAS 98(13) 7611-7616 (2001). cited by other . Kaplitt et al., "Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain," Nature Genetics 8 148-154 (1994). cited by other . Klein et al., "NGF gene transfer to intrinsic basal forebrain neurons increases cholinergic cell size and protects from age-related, spatial memory deficits in middle-aged rats," Elsevier Science B.V., Brain Research 875 144-151 (2000). cited by other . Kordower et al., "Neurodegeneration Prevented by Lentiviral Vecotr Delivery of GDNF in Primate Models of Parkinson's Disease," Science 290 767-772 (Oct. 27, 2000). cited by other . Larsson et al., "Suppression of Insult-Induced Neurogenesis in Adult rat Brain by Brain-Derived Neurotrophic Factor," Experimental Neurology 177 1-8 (2002). cited by other . Luo et al., "Subthalamic GAD Gene Therapy in a Parkinson's Disease Rat Model," Science 298 425-429 (Oct. 11, 2002). cited by other . Manno et al., "AAV-mediated actor IX gene transfer to skeletal muscle in patients with severe hemophilia B," Blood 101(8) 2963-2972 (Apr. 15, 2003). cited by other . McCown et al., "Differential and persistent expression patterns of CNS gene transfer by a adeno-associated virus (AAV) vector," Elsvier Science B.V., Brain Research 99-107 (1996). cited by other . Naldini et al., "Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector," Proc. Natl. Acad. Sc.I. USA 93 11382-11388 (Oct. 1996). cited by other . Xia et al., "the HIV Tat protein transduction domain improves the biodistribution of .beta.-glucuronidase expressed form recombinant viral vectors," Nature Biotechnology 19 640-644 (Jul. 2001). cited by other.
Primary Examiner:	Schnizer; Richard
Attorney, Agent or Firm:	Myers, Bigel, Sibley & Sajovec, P.A.
Parent Case Data:	RELATED APPLICATION INFORMATION This application claims the benefit of U.S. Provisional Application No. 60/376,628; filed Apr. 30, 2002, which is incorporated by reference herein in its entirety.

Claims:

What is claimed is: 1. A delivery vector comprising: a) an adenovirus associated (AAV) vector genome comprising 5' and 3' AAV inverted terminal repeat (ITR) sequences; b) a first nucleotide sequence encoding a polypeptide of interest; and c) a second nucleotide sequence encoding a fibronectin secretory signal sequence that is operatively associated with the first nucleotide sequence of (b), wherein the second nucleotide sequence is selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:14 or SEQ ID NO:15; (ii) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2 or an active fragment thereof having secretory signal activity, SEQ ID NO:5 or an active fragment thereof having secretory signal activity, or SEQ ID NO:6 or an active fragment thereof having secretory signal activity; and a nucleotide sequence that differs from the nucleotide sequence of (ii) above due to the degeneracy of the genetic code, and wherein the second nucleotide sequence does not contain nucleic acids that encode fibronectin amino acid sequence beyond the carboxy terminal side of the cleavage site of the signal sequence. 2. A pharmaceutical composition comprising the delivery vector of claim 1 in a pharmaceutically acceptable carrier. 3. The delivery vector of claim 1, wherein said AAV vector genome does not encode AAV Rep or AAV capsid proteins. 4. The delivery vector of claim 1, wherein said first nucleotide sequence encoding a polypeptide of interest is operatively associated with an inducible promoter. 5. The delivery vector of claim 4, wherein said inducible promoter is selected from the group consisting of a dexamethasone inducible promoter, a tetracycline regulated promoter, an RU486-inducible promoter, an endysone-inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter. 6. The delivery vector of claim 1, wherein said first nucleotide sequence encoding a polypeptide of interest is operatively associated with a promoter that is functional in the central nervous system. 7. The delivery vector of claim 1, wherein said polypeptide of interest is galanin. 8. The delivery vector of claim 1, wherein said polypeptide of interest is selected from the group consisting of galanin, neuropeptide Y, cholecystokinin, thyrotropin-releasing hormone, neurotensin, oxytocin, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell derived growth factor, met-enkephalin, leu-enkephalin, dynorphin, .beta.-endorphin, leptin, a semaphorin peptide, tyrosine hydroxylase, aromatic amino acid decarboxylase, brain-derived neurotrophic factor, nerve growth factor, superoxide dismutase, catalase, glutathione peroxidase, adenosine A-1 receptor, GABA-A receptor, glutamate decarboxylase, neurotrophic factor-3, neurotrophic factor-4 and somatostatin. 9. A method of delivering a nucleic acid to a cell of the central nervous system, comprising contacting the cell with the delivery vector of claim 1 under conditions sufficient for the delivery vector to be introduced into the cell. 10. The method of claim 9, wherein the cell is selected from the group consisting of: (a) a brain cell; b) a neuron, astrocyte, oligodendrocyte, microglial cell, fibroblast, endothelial cell, astroglial cell, or ependymal cell; and (c) a cell from the limbic system, spinal cord, neocortex, thalamus, hypothalamus, epithalamus, pineal gland, corpus striatum, cerebrum, basal ganglia, amygdala, brainstem, cerebrum, cerebellum, striatum, hippocampus, inferior colliculus, pituitary or substantia nigra. 11. The method of claim 9 wherein the delivery vector is formulated in a pharmaceutically acceptable carrier. 12. The method of claim 11, wherein the subject has epilepsy. 13. The method of claim 11, wherein said administering step is carried out by direct injection. 14. The method of claim 11, wherein said administering step is carried out by stereotaxic injection. 15. The method of claim 11, wherein said administering step is to a region of the brain. 16. The method of claim 11, wherein said administering step is to a region selected from the group consisting of the limbic system, spinal cord, neocortex, thalamus, hypothalamus, epithalamus, pineal gland, corpus striatum, cerebrum, basal ganglia, amygdala, brainstem, cerebrum, cerebellum, striatum, hippocampus, pituitary gland and substantia nigra.

Description: